Use of R-(+)-α-lipoic acid, R-(−)-dihydrolipoic acid and metabolites in the form of the free acid or as salts or esters or amides for the preparation of drugs for the treatment of diabetes mellitus as well as of its sequelae

ABSTRACT

The invention relates to the use of R-(+)-α-lipoic acid, R-(−)-dihydrolipoic acid or their metabolites, salts, esters and amides for the synthesis of drugs for the treatment of diabetes mellitus of types I and II, compensated and decompensated insulin resistance and sequelae or late complications of diabetes mellitus, such as cataracts, polyneuropathy, nephropathy, as well as sequelae or late complications of insulin resistance. These drugs mentioned can also be used advantageously in combination with other antidiabetic drugs, particularly with insulin, and/or other additives or stabilizers or adjuvants, such as vitamin B, vitamin C, NADH, NADPH and ubiquinone. 
     The invention furthermore relates to the use of R-(+)-α-lipoic acid, R-(−)-dihydrolipoic acid or their metabolites, as well as their salts, esters and amides for the preparation of drugs for the treatment of diseases with limited function of or a lowered content of the glucose transporters.

This application is a Div. of application Ser. No. 09/285,460 filed Apr.2, 1999, now U.S. Pat. No. 6,117,899, which is a Div. of applicationSer. No. 08/360,924 filed Dec. 21, 1994 now U.S. Pat. No. 5,693,664.

FIELD OF INVENTION

The invention relates to drugs for the treatment of diabetes mellitustypes I and II and its late complications and sequelae or ofsubclinically existing insulin resistance and its late complications andsequelae, as well as to their synthesis.

R-(+)-α-lipoic acid is the physiologically occurring enantiomer of1,2-dithiocyclopentane-3-valeric acid. R-(+)-α-lipoic acid is a coenzymeof α-ketoacid dehydrogenases (pyruvate dehydrogenase, α-ketoglutaratedehydrogenase, etc.) and acts at a key site in the sugar and energymetabolism of the cell. In its function as an intramolecular redoxsystem, it is oxidized (α-lipoic acid) and reduced (dihydrolipoic acid).

The racemate is used as a 50/50 mixture of R-(+)-α-lipoic acid andS-(−)-α-lipoic acid for the treatment of diabetic and alcoholicpolyneuropathy, as well as for the treatment of Amanita phalloidespoisoning and of chronic and alcoholic liver diseases.

It is well known that the pharmacological properties of the enantiomersof α-lipoic acid differ, for example, with respect to theiranti-inflammatory and analgesic effect (European patent EP-A 427 247).It is furthermore reported in the literature that R,S-(+,−)-α-lipoicacid has a blood sugar-lowering effect in the case of alloxan-induceddiabetes in the animal model. In this connection, it has not beenresolved whether this effect due to interference with the secretion ofinsulin or directly due to the activation of the pyruvate dehydrogenase(C. V. Natraj et al., J. Biosci. vol. 6(1), 37-46 (1984)). Metabolicdeviations resulting from diabetes, such as hyperglycemia, ketonemia,ketonuria, reduced glycogen in the tissue and a decreased synthesis offatty acids in the liver are corrected in animal experiments by theadministration of lipoic acid (S. S. Wagh, C. V. Natraj et al., J.Biosci. vol. 11, 59-74 (1987)).

It is furthermore known that oxidative stress is associated with apromoting role in late complications of diabetes and that an adjuvantantioxidant therapy (with thioct acid) can lead to a regression of thelate complications of diabetes (W. Kaehler et al., Innere Medizin 48,(1983) 223-232).

In vitro experiments with thioct acid (material from the Calbiochem Co.(racemate)) have confirmed that it increases the glucose assimilation bymuscles. Time studies show that, contrary to the stimulating effect ofinsulin on glucose assimilation, the effect of thioct acid on ratdiaphragms can be recognized in vitro only after a prolonged incubation.According to Haugaard, the mechanism of action of thioct acid appears tobe unlike that of insulin. Its effect is additive to that of insulin (N.and E. S. Haugaard, Biochim. Biophys. Acta 222 (1970) 582-586). However,in this reference there is also no statement concerning the differenteffects of R- and S-thioct acids. Diabetes mellitus is a disease with aninsulin deficiency or a resistance to the action of insulin(decompensated insulin resistance). Subsequently, numerous metabolicdisorders particularly of the carbohydrate and fat metabolism occur evenin the case of still compensated insulin resistance (reduced effect ofinsulin without clinically manifest diabetes type II). In the long run,these disorders can lead to coma and death. The insulin resistance, aswell as the elevated blood sugar and the impaired fat metabolismparticipate in the development of sequelae and late complications (suchas cataracts, neuropathies, nephropathies). The elevated blood sugar canbe treated by substitution with insulin and, in mild cases, by oralantidiabetic drugs. Up to the present, there has not been a recognized,therapeutic possibility for intervening in the insulin resistanceitself.

A basic disorder in the case of diabetes and insulin resistance lies inthe glucose assimilation by muscle cells. In this connection,particularly within the framework of insulin resistance, it is importantto treat the glucose assimilation not by the administration of insulinor by pharmaceutical drugs stimulating the excretion of insulin, but bymechanisms independent thereof (H. U. Haering, H. Mehnert, Diabetologica36, 176-182, 1993).

The metabolization, within the framework of mitochondrial energymetabolism, necessary after the cellular assimilation of glucose, is afurther, necessary step, particularly in the case of a defective glucoseutilization within the framework of insulin resistance. A key enzyme isthe pyruvate dehydrogenase.

Diabetics show increased glycosilation and oxidation of proteins withcorresponding negative consequences for the patients (Z. Makita. et al.,Science 258, 651-653, 1992).

The finding that specifically R-(+)-α-lipoic acid is suitable for thetreatment of diabetes mellitus and insulin resistance, while theS-(−)-α-lipoic acid practically is not usable for this, is new andunexpected and not inferable by those skilled in the art. Our owninvestigations have shown that, in animal experiments, the key enzyme,pyruvate dehydrogenase, surprisingly was inhibited by the S-(−)-α-lipoicacid.

It is therefore an object of the invention to make available drugs forthe treatment of compensated and decompensated insulin resistance and,with that, of associated diseases and sequelae, or of diabetes mellitusand its sequelae and late complications. The assimilation of blood sugarin the tissue is promoted. This is of clinical relevance in the case ofpathological disorders of the control of blood sugar adjustment, as inthe case of diabetes mellitus types I and II, or in the case ofdisorders in insulin sensitivity of the tissue (insulin resistance).This applies in the case of monotherapy, as well as in the case of acombination with other drugs for the treatment of diabetes mellitus orof insulin resistance, such as oral antidiabetic drugs and, inparticular, insulin. The objective of the treatment can also be asavings in the therapeutically administered insulin or in otherantidiabetic drugs, as well as a lowering in the pathologically elevatedendogenous insulin level. Furthermore, late complications or sequelae ofdiabetes mellitus or of insulin resistance can also be affectedtherapeutically by the treatment of the basic diseases.

Surprisingly, it has now been found that preferably R-(+)-α-lipoic acidproves to be suitable for the treatment of diabetes mellitus types I andII and its sequelae and late complications and for the treatment ofsubclinically and clinically manifest insulin resistance and itssequelae.

Pharmacological Examples

1. Pyruvate Dehydrogenase Activity after Chronic Administration inDifferent Tissues of the Spontaneously Diabetic Rat

Results

Trend after two administrations: Lowered by S-(−)-α-lipoic acid,increased by R-(+)-α-lipoic acid

Description of the Experiment

After the manifestation of the diabetes, spontaneously diabetic rats(BB-Wol BB, of the Moellegard Company, Denmark, n=10/group) received 0.3mL of neutral 0.12 M (corresponding to 50 mg/kg of body weight)R-(+)-α-lipoic acid or S-(−)-α-lipoic acid daily, administered in thevein of the tail. A control group received physiological salt solution.After 7 days, the animals were sacrificed. The pyruvate dehydrogenaseactivity was determined in the heart muscle. The tissue was homogenized.

Measurement of the Pyruvate Dehydrogenase Activity

Test Principle

Pyruvate+NAD⁺+CoA→Acetyl-CoA+CO₂+NADH+H⁺

The extinction of the reduced coenzyme is measured at 339 nm in cuvetteswith a Shimadzu UV 210 Detector at 37° C. The isolation of the enzymecomplex (R. Koeplin, Ph.D. Thesis, University of Tuebingen, FRG, 1988,C. J. Stanley, R. N. Perham, Biochem. J. 191, 147-154, 1980) and theenzyme assay (O. H. Lowry et al. J. Biol. Chem. 256, 815-822, 1951) werecarried out as described. The protein was measured by the method ofLoary (N. Bashan et al., Am. J. Physiol. m262 (Cell Physiol. 31):C682-690, 1992).

2. Glucose Assimilation in Muscle Cells under Insulin (Klip)

Results

Glucose Assimilation

Compared to the S enantiomer, the R enantiomer (2.5 mM) stimulatesglucose assimilation by a factor greater than 2; at the sameconcentration the S enantiomer is less effective.

Glucose Assimilation in Muscle Cells Glucose Assimilation GlucoseGlucose Incubation Control Assimilation Assimilation Time (pmol/mg ×min) R (pmol/mg × min) S (pmol/mg × min) 15 15.1 ± 0.4 16.7 ± 0.6 16.3 ±0.3 30 12.1 ± 0   15.9 ± 0.9 14.8 ± 0.7 60 16.5 ± 0.4 26.1 ± 0.9 21.6 ±0.4 120 15.7 ± 0.6 27.0 ± 0.4 20.5 ± 0.8

Glucose Assimilation in Muscle Cells in Conjunction with Insulin (200nM) R-(+)-α-Liponic Acid (2.5 mM) Glucose Glucose Assimilation GlucoseAssimilation Glucose Insulin + Assimilation R-(+)-α- AssimilationR-(+)-α- Control Liponic Acid Insulin Liponic Acid Incubation (pmol/mg ×(pmol/mg × (pmol/mg × (pmol/mg × Time (min) min) min) min) min) 15 20.0± 0.9 23.2 ± 0.5 24.7 ± 0.9 25.1 ± 0.6 30 18.1 ± 0.6 21.1 ± 0.4 21.6 ±0.4 21.1 ± 0.2 60 18.0 ± 0.6 25.7 ± 0.5 23.7 ± 0.5 26.2 ± 0.7

The effect of the R enantiomer is comparable to that of insulin (200nM); however, the two effects are not additive. In contrast toR-(+)-α-lipoic acid, the S enantiomer decreases the effect of insulin.

Glucose Assimilation in Muscle Cells in Conjunction with Insulin (200nM) S-(−)-α-Liponic Acid, (2.5 mM) Glucose Glucose Assimilation GlucoseAssimilation Glucose Insulin + Assimilation S-(−)α- AssimilationS-(−)-α- Control Liponic Acid Insulin Liponic Acid Incubation (pmol/mg ×(pmol/mg × (pmol/mg × (pmol/mg × Time (min) min) min) min) min) 15 14.5± 0.3 14.8 ± 0.4 17.7 ± 0.3 16.0 ± 0.4 30 13.8 ± 0.5 13.3 ± 0.4 16.3 ±0.5 15.7 ± 0.3 60 15.6 ± 0.5 16.0 ± 0.2 22.3 ± 0.5 19.8 ± 1.1

Description of the Experiment

The tissue muscle cells (L6 myotubes) were prepared in 24-bole platesand differentiated. After incubation with the test substances, an assaywas carried out to determine hexose assimilation (³H-2-desoxyglucose, 10μM, 10 minutes). Insulin was added at a concentration of 200 nM and theα-lipoic acid enantiomers were added at a concentration of 2.5 mM. Afterthe cells were washed and then lysed with NaOH, the radioactivityabsorbed was measured in a counter. Parallel experimental batches werecarried out with cytochalasin-B, in order to determine the glucosetransporter-dependent glucose translocations.

The results can be expressed as pmol/min×mg of protein. The experimentswere carried out by the method described by U.-M Koivisto et al., J.Biol. Chem. 266, 2615-2621, 1991.

4. Effect on the Translocation of Glucose Transporters

Results

R-(+)-α-Lipoic acid stimulates the translocation of glucose transporters(Glut 1 and GLUT 4) from the cytosol to the plasma membrane; this isequivalent to an activation. S-(−)-α-Lipoic acid has no effect or has aninhibiting effect and appears to lower the total content of glucosetransporters in the cell (GLUT4). A translocation of the glucosetransporters corresponds to an activation of the most important cellularglucose assimilation mechanisms. Insulin also stimulates the glucosetransporter translocation.

Effect of Enantiomers of α-Liponic Acid (2.5 mM) on the Translocation ofGLUT1 Glucose Transporters in L6-Myotubes Plasma Membrane LightMicrosomal Treatment (relative units) Fraction (relative units) Control1.00 1.00 R-(+)-Lipoate 1.56 ± 0.25 0.46 ± 0.06 S-(−)-Lipoate 0.93 ±0.37 0.38 ± 0.09 Insulin 1.07 ± 0.14 0.68 ± 0.10

Effect of Enantiomers of α-Liponic Acid (2.5 mM) on the Translocation ofGLUT1 Glucose Transporters in L6-Myotubes Plasma Membrane LightMicrosomal Treatment (relative units) Fraction (relative units) Control1.00 1.00 R-(+)-Lipoate 1.56 ± 0.25 0.46 ± 0.06 S-(−)-Lipoate 0.93 ±0.37 0.38 ± 0.09 Insulin 1.07 ± 0.14 0.68 ± 0.10

Description of the Experiment

L6 myotubes in 15 cm dishes (n=4 to 5) were enlisted and incubated forone hour with 2.5 mM lipoate in MEM with 5 mM of glucose and 2% of fetalbovine serum. The cells were removed, homogenized and worked up infractions (4° C.). The working up was carried out in an HEPES bufferwith a defined addition of protease inhibitor. The cell fractions wereobtained in 6 defined centrifuging steps. The fractions were added to a10% polyacrylamide gel for a Western Blot analysis. The glucosetransporters were determined with anti-GLUT1 and anti-GLUT4 antibodiesusing iodine-labeled protein A and autoradiographic detection.

5. Effect on the Cellular Content of Glucose Transporters

Results

After four hours of incubation, R-(+)-α-lipoic acid increases thecellular content of GLUT1 and GLUT4 glucose transporters. S-(−)-α-Lipoicacid has no effect or lowers the cellular content.

Effect of Liponic Acid Enantiomers (2.5 mM) after 4 Hours of Incubationon the Content of Glucose Transporters in L6-Myotubes GLUT1 (arbitraryGLUT4 (arbitrary Treatment units) units) Control 1.00 1.00 R-(+)-Lipoate1.81 ± 0.01 1.55 ± 0.24 S-(−)-Lipoate 1.08 ± 0.01 0.79 ± 0.47

Description of the Experiment

L6 myotubes were incubated for 4 hours with 2.5 mM lipoic acidenantiomers in an MEM medium with 2% fetal bovine serum and 5 mMglucose. The glucose transporters were detected as described above. Themembrane fraction is obtained after a single centrifugation at 210,000g.

6. Diabetes Induced Tissue Damage

Results

In a diabetes animal model (streptozotocin-induced diabetes), it was nowsurprisingly observed that R-thioct acid corrects numerouspathologically changed parameters (glycosilated hemoglobin, proteinoxidation), whereas the S enantiomer exhibits a lesser effect to noeffect. Surprisingly and additionally, the mortality of the animalgroups in the group exposed to the S enantiomer was increased, while themortality in the group with the R enantiomer was reduced in comparisonto the control.

Glycosilated Hemoglobin Experimental % Glycosilated Group HemoglobinControl 9.7 ± 1.5 (n = 8)  R-Thioct acid diet 8.4 ± 1.3 (n = 11)S-Thioct acid diet 10.7 ± 2.1 (n = 6) 

Glycosilated Hemoglobin Experimental % Glycosilated Group HemoglobinControl 9.7 ± 1.5 (n = 8)  R-Thioct acid diet 8.4 ± 1.3 (n = 11)S-Thioct acid diet 10.7 ± 2.1 (n = 6) 

Glycosilated Hemoglobin Experimental % Glycosilated Group HemoglobinControl 9.7 ± 1.5 (n = 8)  R-Thioct acid diet 8.4 ± 1.3 (n = 11)S-Thioct acid diet 10.7 ± 2.1 (n = 6) 

Description of the Experiment

Thioct acid enantiomers were administered for 14 weeks by mouth,together with the food (1.65 g/kg of food), to female Wistar rats (n=3to 6/group) in separate groups.

In the eighth week, streptozotocin diabetes was induced in the animals.Six weeks after the induction of the diabetes, the surviving animalswere sacrificed. Tissue was taken and analyzed.

R(+)-α-Lipoic acid can thus be regarded a highly specific effective drugfor the treatment of diabetes mellitus types I and II as well as ofdisorders in the insulin sensitivity of the tissue (insulin resistance)and of sequelae and late complications. Moreover, R-(+)-α-lipoic acidcan be used in the case of diseases with a reduced glucose transportercontent or a defective glucose transporter translocation, such ascongenital or hereditary glucose transporter deficiency. Likewise,R-(+)dihydrolipoic acid, the metabolites such as bisnor- andtetranor-lipoic acid and their salts, esters and amides can be used.

The following, for example, come into consideration as indications forthe use of drugs, which contain the materials mentioned:

diabetes mellitus types I and II

subclinically and clinically manifest insulin resistance and theirsequelae (compensated and decompensated insulin resistance)

cataracts

polyneuropathies

nephropathies

glucose transporter deficiency

The R-(+)-α-lipoic acid, R-(−)dihydrolipoic acid or their metabolites(such as bisnor- or tetranor-lipoic acid), as well as their salts,esters, amides are synthesized by known methods (see, for example,German Offenlegungsschrift 41 37 773).

The invention also relates to the use of drugs, which contain theoptically pure R-(+)-α-lipoic acid, R-(−)-dihydrolipoic acid or theirmetabolites as well as their salts, esters and amides, for the treatmentfor the diseases named above.

Pharmaceutical Examples

The amounts by weight, given in the patent, relate in each case to thepure optical isomer and not to the salts. When salts, esters or amidesare used, the weights must be adapted correspondingly to the changedmolecular weights.

The salts are synthesized by known methods (see also Patent EP-A901213405). The pharmaceutical preparations generally contain 3 to 5 mgof the compounds used pursuant to the invention as a single dose. Afterrepeated administrations, the effective level attained in the bodyshould be between 0.1 and 100 mg/kg of body weight.

The material is administered in the form of tablets, chewable tablets,sucking tablets, pills, capsules, granulates, coated tablets,effervescent tablets, effervescent powders, finished drink solutions,liquid forms for parenteral administration and aerosols. Finished drinksolutions and liquid forms for parenteral administration can bealcoholic or aqueous solutions, suspensions and emulsions.

Preferred embodiments are, for example, tablets, which contain between10 mg and 2 g of active substance, as well as solutions, which containthe active substance in amounts of between 1 mg and 200 mg per mL ofliquid.

The following may be named as single doses of the active ingredient:

a. oral forms: 10 mg to 3 g

b. parenteral forms (intravenous or intramuscular): 10 mg to 12 g

c. inhalants: 10 mg to 2 g.

The doses a) to c) can be administered, for example, 1 to 6 times dailyor as an intravenous drip.

EMBODIMENTS Example 1

Tablets with 100 mg of R-(+)-α-Lipoic Acid

R-(+)-α-Lipoic acid (250 g) is triturated uniformly with 750 g ofmicrocrystalline cellulose. After the mixture is screened, 250 g ofstarch (Starch 1500/Colorcon), 732.5 g of lactose, 15 g of magnesiumstearate and 2.5 g of highly dispersed silica are admixed and themixture is pressed into tablets weighing 800.0 mg. One tablet contains100 mg of R-(+)-α-lipoic acid. If necessary, the tablets can be coatedin a conventional manner with a film, which is soluble or permeable togastric juices.

Example 2

Ampules with 250 mg of R-(+)-α-Lipoic Acid as Trometamol Salt in 10 mLof Injection Solution

R-(+)-α-Lipoic acid (250 g), together with 352,3 g of trometamol(2-amino-2-(hydroxymethyl)-1,3-propylene glycol) is dissolved withstirring in a mixture of 9 liters of water for injection purposes and200 g of 1,2-propylene glycol. The solution is made up to 10 liters withwater for injection purposes and subsequently filtered through a glassfiber prefilter and then through a membrane filter with a pore size of0.2 μm. The filtrate (10 mL amounts) is filled under aseptic conditionsinto 10 mL ampules. In 10 mL of injection solution, 1 ampule contains250 mg of R-(+)-α-lipoic acid as the trometamol salt.

Example 3

Ampules with 250 mg of R-(−)-Dihydrolipoic Acid in 10 mL of InjectionSolution

Trometamol (60 mg) and 1 g of the disodium salt ofethylenediaminetetraacetic acid are dissolved in 1.8 liters of water forinjection purposes. Nitrogen is bubbled for 30 minutes through thesolution. While the bubbling of nitrogen is continued, 2 g of sodiumdisulfite and subsequently 50 g of R-(−)-dihydrolipoic acid aredissolved in the mixture. The solution is made up to a volume of 2liters with water for injection purposes, through which nitrogen hasbeen bubbled. After careful mixing, the solution is filtered through amembrane filter with a pore size of 0.2 μm and the filtrate is filledinto 10 mL ampules under aseptic conditions, nitrogen being bubbledthrough the filtrate before and after it is filled into the ampules. Oneampule contains 250 mg of R-(−)-dihydrolipoic acid as the trometamolsalt in 10 mL of solution.

What is claimed is:
 1. A process for the treatment of sequelae and latecomplications of diabetes mellitus comprising administering to a patientan effective amount of pure R-(+)-α-lipoic acid, pureR-(−)-dihydrolipoic acid, amides, salts, metabolites or esters thereof.2. The process of claim 1 further comprising the administration of atleast one substance selected from the group comprising vitamin E,vitamin C, NADH, NADPH and ubiquinone as additives, stabilizers oradjuvants.